THE EFFECTS OF CELL AGEING ON METABOLISM IN RAINBOW TROUT (ONCORHYNCHUS MYKISS) RED BLOOD CELLS

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1 The Journl of Experimentl Biology 23, (2) Printed in Gret Britin The Compny of Biologists Limited 2 JEB THE EFFECTS OF CELL AGEING ON METABOLISM IN RAINBOW TROUT (ONCORHYNCHUS MYKISS) RED BLOOD CELLS M. C. L. PHILLIPS, C. D. MOYES AND B. L. TUFTS Deprtment of Biology, Queen s University, Kingston, Ontrio, Cnd K7L 3N6 Author for correspondence (e-mil: tuftsb@biology.queensu.c) Accepted 14 Jnury; published on WWW 21 Februry 2 The effects of cell ge on metbolism in the nucleted red blood cells of rinbow trout (Oncorhynchus mykiss) were exmined. Red blood cells were seprted ccording to ge using fixed-ngle centrifugtion. The men erythrocyte hemoglobin concentrtion in old red blood cells ws found to be 12 % of tht in young red blood cells. In young red blood cells, the ctivities of the mitochondril enzymes citrte synthse nd cytochrome oxidse were %, respectively, of those mesured in old red blood cells. The ctivity of the glycolytic enzyme lctte dehydrogense in young red blood cells ws 17 % of tht in old red blood cells, wheres the ctivity of the glycolytic enzyme pyruvte kinse ws not significntly ffected by cell ge. In ddition, Summry young red blood cells consumed over twice s much O 2 nd devoted 5 % more O 2 to protein synthesis nd the ctivity of N + /K + -ATPse thn old red blood cells. Red blood cell ge did not significntly ffect the rte of lctte production. This study shows tht geing in rinbow trout nucleted red blood cells is ccompnied by significnt decline in erobic energy production nd the processes it supports, s well s corresponding increse in the glycolytic contribution to metbolism. Key words: rinbow trout, Oncorhynchus mykiss, metbolism, geing, fish, erythrocyte, red blood cell. Introduction Striking differences in morphology exist between the red blood cells of mmmlin nd non-mmmlin vertebrtes. One of the most notble differences is tht mmmlin red blood cells lose their nuclei before entering the blood, wheres non-mmmlin red blood cells remin nucleted throughout their circultory lifespn (Sekhon nd Bems, 1969; Boutilier nd Ferguson, 1989). Mmmlin red blood cells re lso typiclly biconcve nd extrude their orgnelles shortly fter relese into the circultion, wheres non-mmmlin red blood cells re biconvex nd retin significnt number of orgnelles nd ribosomes while in the blood (Sekhon nd Bems, 1969; Lne nd Thrp, 198; Lne et l., 1982). In contrst to their enucleted counterprts, the retention of orgnelles such s mitochondri enbles nucleted red blood cells to utilize erobic metbolism (Boutilier nd Ferguson, 1989). This erobic ATP production my be required for processes, such s protein synthesis (Speckner et l., 1989; Keen et l., 1989), tht re bsent from enucleted red blood cells. To dte, however, the functionl significnce of the dditionl erobic cpcity of nucleted red blood cells hs not been thoroughly investigted. The effects of cell geing on energy producing nd utilizing pthwys in nucleted red blood cells re lso unknown (Wlsh et l., 1998). The loss of mitochondri with geing described in ultrstructurl studies (Sekhon nd Bems, 1969; Keen et l., 1989) suggests significnt reduction in the cpcity to produce ATP erobiclly. It is not known, however, whether these chnges reflect n overll reduction in totl (oxidtive nd glycolytic) ATP production or whether incresed glycolytic ATP production might compenste for decline in erobic energy production s nucleted red blood cells ge. Reductions in polyribosome content (Lne nd Thrp, 198; Keen et l., 1989) lso provide evidence tht energy utiliztion for protein synthesis declines during geing in nucleted red blood cells. Assuming tht totl energy production does decline with ge, chnges in energy utiliztion pthwys, such s tht for protein synthesis, would be expected since energy utiliztion must be mtched to energy production under stedy-stte conditions. At present, however, the effects of geing on specific energy utiliztion pthwys in nucleted red blood cells hve not been well described. On this bckground, the purpose of the present study is to exmine the effects of cell ge on erobic nd glycolytic metbolism, s well s the energy requirements of protein synthesis nd the N + /K + -ATPse, in the nucleted red blood cells of rinbow trout Oncorhynchus mykiss. It is hypothesized tht geing will be ssocited with decline in the rte of respirtion nd possibly compenstory increse in glycolysis. Moreover, ny reductions in totl energy production will be mtched by declines in energy-consuming processes.

2 14 M. C. L. PHILLIPS, C. D. MOYES AND B. L. TUFTS Mterils nd methods Blood preprtion Freshwter rinbow trout Oncorhynchus mykiss (Wlbum) (1 4 kg) were obtined from commercil fish htchery (Pure Springs Trout Frm, Belleville, Ontrio, Cnd) nd held in the Animl Cre Fcility t Queen s University, Kingston, Ontrio. Fish were kept in erted dechlorinted wter t 1 15 C nd fed commercil fish pellets t regulr intervls of 3 4 dys. Individul trout were nesthetized in dechlorinted wter contining 25 mg l 1 tricine methne sulphonte (MS-222; Sigm) nd 5 mg l 1 NHCO 3 buffer. Blood (pproximtely 15 ml) ws drwn into syringe vi cudl puncture nd plced in chilled flsk contining heprinized (4 i.u. ml 1 ) sline (in mmol l 1 : 124 NCl, 5 KCl,.5 MgCl 2, 1.1 CCl 2, 5.5 glucose, 1 NHCO 3 ). The red blood cells were wshed three times with sline, nd specil cre ws tken to remove ll white blood cells using n spirtor. Seprtion of ge frctions Following the wshing procedure, red blood cells were seprted into different ge frctions using fixed-ngle centrifugtion. This technique hs been used successfully in previous studies (Murphy, 1973; Cohen et l., 1976; Speckner et l., 1989), nd only minor modifictions were incorported into the present experiments. Initilly, red blood cells were djusted to hemtocrit of 8 % nd filled to the top of nrrow centrifuge tube (dimeter 13 mm, length 1 mm, volume 6.5 ml). The red blood cells were then centrifuged (1 g, temperture 14 C) for 15 min with JA-2 rotor inside centrifuge (Beckmn; model J2-21M) t fixed ngle of 3 to the verticl. Following centrifugtion, six equl 1 ml volumes of red blood cells were crefully removed from the tube using pipette. Ech 1 ml frction ws numbered ccording to its position in the centrifuge tube (frction 1 for red blood cells t the top, frction 6 for red blood cells t the bottom). Individul frctions were then wshed twice in sline nd kept on ice for subsequent experiments. Experimentl protocol nd nlyses The protocol consisted of four seprte series of in vitro experiments. In the first series, the men erythrocyte hemoglobin (Hb) concentrtion (MEHC) ws mesured in frctions 1 6 for vrying centrifugtion speeds nd times to obtin the best ge seprtion possible. In the second series, the ctivities of the metbolic enzymes citrte synthse (CS), cytochrome oxidse (COX), pyruvte kinse (PK) nd lctte dehydrogense (LDH) were mesured in frctions 1 6. In the third series, O 2 consumption ws mesured for frctions 1 6, nd inhibition experiments with cycloheximide nd oubin were conducted on frctions 1 nd 6 only. In the fourth series, lctte production ws mesured for frctions 1 nd 6 only. Hemoglobin The Hb content of ech frction ws determined using the Hb-cynide method (Betke nd Svelsberg, 195; vn Kmpen nd Zijlstr, 1961) nd DU 64 spectrophotometer (Beckmn). The hemtocrit (Hct) of ech frction ws mesured fter centrifuging the smples for 4 min in n IEC MB microhemtocrit centrifuge (Dmon/IEC Division). MEHC for ech frction ws clculted s [Hb]/Hct. Pyruvte kinse, lctte dehydrogense nd citrte synthse Enzyme smples were prepred for ech ge frction by dding 1 µl of pcked red blood cells to 1 ml of cell extrction buffer (2 mmol l 1 Hepes, ph 7., 1 mmol l 1 EDTA,.1 % Triton X-1) followed by vortexing nd soniction for severl seconds t low setting. Smples were frozen t 8 C until the dy of the ssy. PK ctivity ws mesured t 25 C using Spectrmx Plus plte spectrophotometer (Moleculr Devices) nd detected t 34 nm s the rte of disppernce of NADH from 3 µl of mixture contining 5 mmol l 1 Hepes, ph 7., 5 mmol l 1 ADP, 1 mmol l 1 KCl, 1 mmol l 1 MgCl 2,.15 mmol l 1 NADH, 1 µmol l 1 fructose 1,6-biphosphte, 5 mmol l 1 phosphoenolpyruvte nd 5 units of LDH. The ctivity of LDH ws determined t 25 C s the rte of removl of NADH from 3 µl of mixture contining 2 mmol l 1 Hepes, ph 7.,.15 mmol l 1 NADH nd.5 mmol l 1 pyruvte. CS ctivity ws mesured t 15 C using DU 64 spectrophotometer (Beckmn) nd detected t 412 nm s the rte of formtion of 5,5 -dithiobis-(2 -nitrobenzoic cid) (DTNB) in 1 ml of mixture contining 2 mmol l 1 Tris bse,.1 mmol l 1 DTNB,.12 mmol l 1 cetyl coenzyme A nd.2 mmol l 1 oxlocette. Non-specific thiolse ctivity for CS, determined in the bsence of oxlocette, ws subtrcted from the rte in the complete mixture. The controls for both PK (bsence of phosphoenolpyruvte) nd LDH (bsence of pyruvte) displyed no detectble ctivities. Cytochrome oxidse Pcked red blood cells (2 µl) from ech frction were suspended in 8 µl of sline, sonicted (2 s, low setting) nd centrifuged for 5 min t 1 g in MicroMx centrifuge (IEC). The resulting nucler pellet ws discrded, nd the superntnt ws centrifuged for n dditionl 1 min t 1 g to isolte the mitochondril pellet, which ws then resuspended in 2 µl of cell extrction buffer. COX ws ssyed t 25 C nd 55 nm using Spectrmx Plus plte spectrophotometer (Moleculr Devices). The specific ctivity of ech frction ws mesured by detecting the rte of reduction of cytochrome c in 2 µl of mixture contining 5 mmol l 1 Tris bse, 5 µmol l 1 cytochrome c nd.5 % Tween 2. COX ctivities were expressed on the bsis of the strting volumes of blood. Rtes of O 2 consumption Blood from ech of the six ge frctions (2 ml t Hct of 2 %) ws equilibrted with humidified ir t 15 C in gently rotting erted flsk for 1 h. Following the equilibrtion period, 6 µl smple of blood ws dded to n ir-tight

3 Effects of geing on trout red blood cell metbolism 141 chmber fitted with n E546 P O electrode (Rdiometer) nd gently stirred for 12 min t 15 C. During this time, the chnge in O 2 prtil pressure (P O ) of ech frction ws mesured with n OM2 O 2 nlyzer (Cmeron Instrument Compny) nd recorded with n Omniscribe D-5 physiogrph chrt recorder (Allen Dtgrph). As described by Wood et l. (199), preliminry experiments showed tht Hb remined fully sturted during the mesurement period nd, therefore, tht the chnge in P O represented red blood cell O 2 consumption from the surrounding medium rther thn the removl of O 2 from Hb. The reltive percentge of O 2 consumption required for protein synthesis ws determined by compring O 2 consumption in the presence nd bsence of cycloheximide t finl concentrtion of 1 µgml 1 (Currie nd Tufts, 1997). The reltive percentge of O 2 consumption required for the N + /K + -ATPse ws lso determined by compring O 2 consumption in the presence nd bsence of oubin t finl concentrtion of.1 mmol l 1 (Tufts nd Boutilier, 1991). An O 2 solubility constnt of µmol l 1 mmhg 1 (1 mmhg=.133 kp; Boutilier et l., 1984) ws used to clculte the rte of O 2 consumption from chnges in P O. Lctte production Blood from frctions 1 nd 6 (4 ml t Hct of 2 %) ws equilibrted with humidified ir in gently rotting erted flsk for 1 h t 15 C. Following the initil equilibrtion period, 2 ml of blood from ech frction ws treted with humidified N 2 for 2 h to induce noxi, while the remining 2 ml continued to be equilibrted with humidified ir for 2 h. Blood smples of 1 ml were tken for both frctions fter the 1 h equilibrtion period nd t the end of the 2 h ertion/noxic period to be cidified in preprtion for the lctte ssy. Smples were cidified with.1 ml of 7 % perchloric cid nd centrifuged for 5 min t 14 g. The superntnt ws neutrlized by dding.3 ml of sturted Tris bse followed by.2 ml of 2 mol l 1 KOH nd then centrifuged gin for 1 min t 14 g. Superntnts were frozen t 8 C until the dy of the ssy. Lctte concentrtions were determined using the hydrzine sink method. Briefly, 15 µl of smple ws dded to 15 µl of ssy mixture (4 mmol l 1 hydrzine, 1 mol l 1 glycine, 2 mmol l 1 EDTA, 4 mmol l 1 NAD +, 2 units of LDH, ph 9.5), nd the rection ws followed for 4 min to completion. Lctte stndrds of vrying known concentrtions were lso run in prllel during this time. The lctte concentrtions of both smples nd stndrds were ssyed in triplicte t 34 nm with Spectrmx Plus plte spectrophotometer (Moleculr Devices). The lctte concentrtions mesured fter the 1 h equilibrtion period were subtrcted from those mesured fter the 2 h intervl of ertion/noxi to eliminte strting concentrtions. Sttisticl nlyses Significnt differences (P.5) between frction mens were detected using one-wy nlyses of vrince (ANOVAs) nd identified using the Student Newmn Keuls test. In experiments involving only frctions 1 nd 6, significnt differences (P.5) between frction mens were detected using pired two-tiled t-tests. Results Men erythrocyte hemoglobin concentrtion MEHC incresed t firly constnt rte with frction ge (Fig. 1); frctions 4 6 ll displyed significntly higher MEHCs thn frction 1, nd the MEHC of frction 6 ws lso significntly greter thn tht of frctions 2 nd 3. The frction 6 men MEHC of 25.9 µg µl 1 red blood cells ws roughly 12 % of the frction 1 men MEHC of µg µl 1 red blood cells. Metbolic enzymes Metbolic enzyme ctivity decresed with red blood cell ge for CS, COX nd LDH, with men frction 1 ctivities rnging from 135 to 2 % of those in frction 6. Mitochondril enzymes (citrte synthse, cytochrome oxidse) CS ctivity fell shrply between frctions 1 nd 2, but remined firly constnt cross frctions 2 6 (Fig. 2A). The men frction 1 CS ctivity of 1.75 nmol µl 1 red blood cells min 1 ws higher thn tht of ny other frction nd ws 135 % of the men frction 6 CS ctivity. Unlike CS, COX ctivity decresed t more constnt rte cross the ge frctions (Fig. 2B). The men frction 1 COX ctivity of.6 nmol µl 1 red blood cells min 1 ws greter thn tht in ny other frction nd over twice s high s the men frction 6 COX ctivity. Thus, the decrese in COX ctivity between frctions 1 nd 6 ws lrger in mgnitude thn the decrese in CS ctivity between the sme two frctions. Glycolytic enzymes (lctte dehydrogense, pyruvte kinse) LDH ctivity peked t 133 nmol µl 1 red blood cells min 1 MEHC (µg µl -1 red blood cells) Red blood cell ge frction Fig. 1. Men erythrocyte hemoglobin concentrtion (MEHC) in six ge frctions of rinbow trout red blood cells. The youngest red blood cells re in frction 1, nd the oldest red blood cells re in frction 6. The letters, b nd c denote vlues tht re significntly different from those for frction 1, 2 or 3 (P.5, N=6), respectively. All vlues re expressed s mens ± S.E.M.,b,c

4 142 M. C. L. PHILLIPS, C. D. MOYES AND B. L. TUFTS Fig. 2. Activities of the metbolic enzymes citrte synthse (CS) (A), cytochrome oxidse (COX) (B), lctte dehydrogense (LDH) (C) nd pyruvte kinse (PK) (D) in six ge frctions of rinbow trout red blood cells (rbcs). The youngest red blood cells re in frction 1, nd the oldest red blood cells re in frction 6. The letters nd b denote vlues tht re significntly different from those for frction 1 nd 2 (P.5, N=6), respectively. All vlues re expressed s mens ± S.E.M. CS ctivity COX ctivity ,b,b,b A B,b LDH ctivity PK ctivity Red blood cell ge frction ,b,b,b C,b D in frction 1 nd decresed shrply in the younger frctions, but levelled off in the older frctions (Fig. 2C). The men frction 1 LDH ctivity ws 17 % of the men frction 6 LDH ctivity. PK ctivity ws not significntly ffected by frction ge (Fig. 2D). However, the men frction 1 PK ctivity of nmol µl 1 red blood cells min 1 ws roughly 13 % of the men frction 6 PK ctivity. Rtes of O 2 consumption The rte of O 2 consumption decresed significntly with frction ge (Fig. 3). The men frction 1 O 2 rte of consumption ws.193 nmol O 2 µl 1 red blood cells min 1, higher thn tht in ny other frction nd over twice s high s the men frction 6 rte of O 2 consumption. The difference in Rte of O2 consumption (nmol O2 µl -1 rbcs min -1 ) Fig. 3. Rtes of O 2 consumption in six ge frctions of rinbow trout red blood cells (rbcs). The youngest red blood cells re in frction 1, nd the oldest red blood cells re in frction 6. The letter denotes vlues tht re significntly different from tht for frction 1 (P.5, N=6). All vlues re expressed s mens ± S.E.M.,b Red blood cell ge frction rtes of O 2 consumption between frctions 1 nd 6 ws similr in mgnitude to the decrese in COX ctivity observed for the sme two ge frctions. Exposure of frctions 1 nd 6 to the respirtory inhibitors cycloheximide or oubin resulted in decreses in rtes of O 2 consumption for both frctions in comprison with uninhibited rtes (Fig. 4). Proportionlly, the decrese in the rte of O 2 consumption from the uninhibited rtes did not differ between frctions 1 nd 6 for either cycloheximide (frction 1 decresed by 26.4 % compred with 27. % for frction 6) or oubin (frction 1 decresed by 36.6 % compred with 4.1 % for frction 6). However, differences in the totl O 2 requirements of both protein synthesis nd the N + /K + -ATPse were observed (Fig. 5). Inhibition of protein synthesis with cycloheximide cused the men frction 1 rte of O 2 consumption to decrese by.6 nmol O 2 µl 1 red blood cells min 1 compred with reduction of only.37 nmol O 2 µl 1 red blood cells min 1 for frction 6. Inhibition of the N + /K + -ATPse with oubin cused the men frction 1 rte of O 2 consumption to fll by.115 nmol O 2 µl 1 red blood cells min 1, while the rte for frction 6 fell by only.67 nmol O 2 µl 1 red blood cells min 1. Thus, frction 1 red blood cells devoted pproximtely 5 % more O 2 consumption towrds protein synthesis nd the N + /K + -ATPse thn frction 6 red blood cells. Lctte production The red blood cells in frctions 1 nd 6 both significntly incresed their rte of lctte production under noxic conditions compred with those under erted conditions (Fig. 6). However, no significnt differences were observed between frctions 1 nd 6 with respect to either erted or noxic rtes of lctte production.

5 Effects of geing on trout red blood cell metbolism 143 Rte of O2 consumption (nmol O2 µl -1 rbcs min -1 ) A B 1 6 Frction 1 6 Frction Control Cycloheximide Control Oubin Fig. 4. Effects of the respirtory inhibitors cycloheximide (A) nd oubin (B) on the rte of O 2 consumption of young (frction 1) nd old (frction 6) rinbow trout red blood cells (rbcs). Asterisks denote significnt difference from the vlue for uninhibited controls for both frctions 1 nd 6 (P.5, N=4). No significnt differences in the rte of O 2 consumption exist between ge groups s proportion of control rtes. All vlues re expressed s mens + S.E.M. Discussion Vertebrte red blood cells cn be seprted into frctions of different ges using fixed-ngle centrifugtion (Murphy, 1973; Cohen et l., 1976; Speckner et l., 1989). This technique relies on density differences between young nd old cells which result lrgely from n increse in MEHC during mturtion (Tooze nd Dvies, 1963; Härdig, 1978; Keen et l., 1989). The mgnitude of the chnge in MEHC observed cross the six cell frctions in the present study (Fig. 1) ws similr to tht in previous studies (Speckner et l., 1989). These results therefore confirm tht the methodology used in this study ws effective in seprting trout red blood cells into frctions with different men ges. Severl other spects of this methodology lso wrrnt discussion. First, previous studies hve shown tht some degree of contmintion between ge frctions should be expected when cells re seprted using this pproch (Piomelli et l., 1967; Beutler, 1985). Thus, the true mgnitude of the differences between ge frctions will probbly be even greter thn tht mesured. Moreover, the mgnitude of the differences between young nd old cells will lso be influenced by the number of frctions chosen for exmintion. Six frctions were chosen for exmintion in the present study. This represented the mximum number of frctions tht still provided n dequte volume of cells for the nlyses. O2 requirement (nmol O2 µl -1 rbcs min -1 ) Frction 1 Frction 6 Protein synthesis N + /K + -ATPse Fig. 5. The O 2 consumption requirements for protein synthesis nd N + /K + -ATPse in young (frction 1) nd old (frction 6) rinbow trout red blood cells (rbcs). Asterisks denote significnt difference from the vlue for frction 1 (P.5, N=4). All vlues re expressed s mens + S.E.M. Nonetheless, it would be expected tht the mgnitude of ny observed differences between the youngest nd oldest cell frctions might hve been even lrger hd the cells been seprted into greter number of frctions. Lstly, it is noteworthy tht we hve shown tht the concentrtion of totl DNA does not vry between ge frctions (S. G. Lund, M. C. L. Phillips, C. D. Moyes nd B. L. Tufts, in preprtion). Thus, the differences observed between the ge frctions do not reflect significnt differences in the number of cells per unit volume between frctions. As documented in numerous previous studies using this pproch, the differences observed between frctions in the present study therefore represent the impct of geing on red blood cell vribles. It is well known tht loss of orgnelles ccompnies nucleted red blood cell geing (Sekhon nd Bems, 1969; Lne et l., 1982; Keen et l., 1989). However, the metbolic Rte of lctte production (nmol lctte µl -1 rbcs min -1 ) Aerted Anoxic Fig. 6. Rtes of lctte production in young (frction 1) nd old (frction 6) rinbow trout red blood cells (rbcs) under conditions of ertion nd noxi. Asterisks denote significnt difference between noxic nd erted rtes of lctte production for both frctions 1 nd 6 (P.5, N=4). No significnt differences exist between frction 1 nd 6 erted or noxic rtes of lctte production. All vlues re expressed s mens + S.E.M. Frction 1 Frction 6

6 144 M. C. L. PHILLIPS, C. D. MOYES AND B. L. TUFTS impct of degrdtion in orgnelles such s mitochondri is unknown. In the present study, the rte of O 2 consumption ws found to decline by t lest 5 % in geing rinbow trout red blood cells (Fig. 3). The underlying bsis for this chnge ppers to be due to coincident chnges in both mitochondril properties nd the cellulr demnds for energy. From mitochondril perspective, CS nd COX ctivity mesurements show tht geing in rinbow trout red blood cells is ssocited with significnt reduction in erobic metbolic cpcity. The CS ctivity in frction 1 is 135 % of tht in ny other frction (Fig. 2A), suggesting tht only the youngest cells hve n elevted cpcity for tricrboxylic cid cycle ctivity. Unlike CS, the ctivity of COX decreses t more grdul rte in geing red blood cells (Fig. 2B), nd the decline in ctivity (5 %) between frctions 1 nd 6 is lrger. The different trends observed between CS nd COX ctivity re interesting for number of resons. First, these results suggest tht cell geing my be ccompnied by chnges in mitochondril structure s well s decresed mitochondril bundnce. Specificlly, CS is loclized in the mitochondril mtrix (i.e. mrker for mitochondril volume) nd COX is loclized in the mitochondril inner membrne (i.e. mrker for criste surfce re). These observtions re lso supported by studies which show tht mitochondril degrdtion in geing red blood cells is chrcterized by symptoms such s swelling nd criste disorgniztion (Sekhon nd Bems, 1969; Keen et l., 1989). Second, the mgnitude of the decline in COX ctivity is very similr to tht observed for O 2 consumption, which suggests tht COX ctivity my be more ccurte indictor of ctul erobic flux thn is CS ctivity. From functionl perspective, young nd old red blood cells devote roughly the sme proportion of the O 2 they consume to protein synthesis (Fig. 4A). Protein synthesis is energeticlly expensive nd my ccount for up to 4 % of whole-niml oxygen consumption in fish (Lyndon et l., 1992). In the present study, protein synthesis ccounts for 26 % of the oxygen consumption of young red blood cells nd 27 % of the oxygen consumption of old cells. However, the bsolute O 2 consumption devoted to protein synthesis in young red blood cells is pproximtely 5 % greter thn tht in old red blood cells (Fig. 5). Younger red blood cells contin more polyribosomes thn old red blood cells (Lne nd Thrp, 198) nd hve higher rte of oxygen consumption thn older red blood cells, so it is not surprising tht they devote more totl O 2 to protein synthesis. Similr trends were observed for the energy requirements of the N + /K + -ATPse. Previous studies on N + /K + -ATPse inhibition show tht it ccounts for 2 25 % of the totl red blood cell O 2 consumption in rinbow trout red blood cells (Tufts nd Boutilier, 1991; Wng et l., 1994). In our experiments, the N + /K + -ATPse ccounted for greter proportion of the totl oxygen consumption; 37 % nd 4 % in young nd old cells respectively (Fig. 4B). The reson tht our current vlues re somewht higher thn those of previous studies is not entirely cler, lthough it is noteworthy tht vrying degrees of drenergic stimultion my contribute to these differences (Tufts nd Boutilier, 1991). As observed for protein synthesis, the bsolute O 2 consumption devoted to the N + /K + -ATPse in young red blood cells is pproximtely 5 % greter thn tht in old red blood cells (Fig. 5). As result of the ccumultion of C 2+, geing red blood cells experience disruption in membrne symmetry (Schwrtz et l., 1985). Since N + /K + -ATPse ctivity is dependent upon the symmetry of the phospholipid environment (Roelofsen, 1981; Deuticke nd Hest, 1987), this my explin why the O 2 requirements of the N + /K + -ATPse lso decrese with cell ge. The lterntive route of ATP production, glycolysis, becomes incresingly importnt s nucleted red blood cells ge. Since lctte production is unffected by red blood cell ge (Fig. 6), wheres the rte of O 2 consumption decreses s red blood cells ge, higher proportion of the totl ATP produced in old red blood cells is derived through glycolysis. Shifts in the reltive importnce of erobic nd nerobic metbolism re known to occur in other instnces, such s during myogenesis in mouse C2C12 myoblsts (Lery et l., 1998). In ddition, previous studies on rinbow trout red blood cells hve shown tht nerobic metbolism plys significnt role in red blood cell energy production nd cn ccount for up to 28 % of cell metbolism even when O 2 is present (Wlsh et l., 199). It is interesting tht the decline in LDH ctivity (Fig. 2C) does not pper to hve negtive effect on glycolytic flux. The functionl significnce of n pprent surplus of LDH ctivity in young compred with old cells is therefore uncler. By mintining higher concentrtion of LDH, young red blood cells my be ble to respond more quickly to conditions of hypoxi or noxi. Alterntively, difference in LDH ctivity between young nd old cells my reflect different bilities to use lctte s n oxidtive fuel. In conclusion, this study shows tht geing in rinbow trout nucleted red blood cells is ccompnied by significnt chnges in metbolism tht re t lest s lrge s those recorded nd re probbly even greter. Young red blood cells consume t lest twice s much O 2 s old red blood cells. The underlying resons for this difference pper to be mitochondril degrdtion, s indicted by reduced CS nd COX ctivity, nd lower cellulr energy demnds, s indicted by decline in the proportion of O 2 consumption devoted to protein synthesis nd N + /K + -ATPse ionoregultion. Since the rte of O 2 consumption in the geing red blood cell decreses, wheres lctte production remins constnt, the role of glycolysis becomes incresingly importnt with cell ge. Finncil support for this study ws provided by Queen s Grdute Fellowship to M.C.L.P. nd NSERC Operting Grnts to C.D.M. nd B.L.T. References Betke, K. nd Svelsberg, W. (195). Stufenphotometrische Hämoglobinstimmung mittels Cynhämoglobin. Biochem. Z. 32, Beutler, E. (1985). Annottion. How do red cell enzymes ge? A new perspective. Br. J. Hemtol. 64,

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